Up-regulated expression of a novel gene in activated human peripheral blood mononuclear cells that is a truncated paralog of the human system l-amino acid transporter 1

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Abstract

Introduction and aims: The human system l-amino acid transporter1 (hLAT1) is one of the CD98 light chains and its gene has been mapped to chromosome 16q24. Our preliminary findings have indicated that in HeLa S3 cells there are transcripts whose nucleotide sequences are very similar but not identical to that of the amino acid transporter. This study intends to examine whether these novel transcripts have biological significance through elucidating their genetic aspects and expression profiles in human cells. Methods: The expression levels of the transcripts were quantified by real-time PCR analysis. Chromosomal mapping of the gene was performed by fluorescence in situ hybridization (FISH). Results: Three types of transcripts were identified and their nucleotide sequences were aligned with the chromosome 16p12 clone with high identity. They encoded 180- or 190-amino acid proteins, showing 92–94% of amino acid identity to the amino-terminal region of the hLAT1 (507 amino acids). However, their 3′ non-coding sequences did not show homology to the nucleotide sequence of the amino acid transporter. Their genes were mapped to chromosome 16p11.2–p13.1 as low-copy repeats (LCRs). The transcription of one of these genes in peripheral blood mononuclear cells was significantly up-regulated when the cells were stimulated with concanavalin A. Conclusion: We have characterized the three truncated paralogs of the hLAT1 gene. It is suggested that the expression of one of these paralogs may play an important role in the activation of peripheral blood mononuclear cells.

Introduction

Fusion regulatory protein-1 (FRP-1) is a glycosylated 80-kDa protein expressed in most cell lines of human origin [1], [2]. Monoclonal antibodies against the FRP-1 regulate cell fusion induced by viruses including Newcastle disease virus, measles virus and human immunodeficiency virus [1], [2], [3]. Furthermore, human peripheral blood monocytes can be fused when incubated in the presence of the monoclonal antibodies and the resulting polykaryocytes exhibit osteoclast-like cell phenotypes [3], [4], [5]. On the other hand, human CD98 is a disulfide-linked complex composed of a glycosylated heavy chain (CD98HC, 85 kDa) and a non-glycosylated light chain (CD98LC, 41 kDa) [6]. Previously, we identified the FRP-1 (80 kDa polypeptide) to be the same as the CD98HC molecule [7]. The CD98HC is a type II transmembrane (TM) glycoprotein with 529 amino acids [8], [9], whose biological functions other than the fusion regulation have been proposed [3], [10]. For example, the CD98HC functions as an amino acid transporter [11] or an Na+/Ca2+ exchanger [12], [13]. Furthermore, the CD98HC has been reported as an important regulator of integrin-mediated cell adhesion [4], [14], [15]: the CD98HC associates with the integrin β1A possibly via the cytoplasmic domains [16].

In contrast to CD98HC, the structure and function of the CD98LC have long been unresolved. Recently, we have purified CD98LC polypeptide from HeLa S3 cells and determined its N-terminal amino acid sequence [17]. Subsequently, a full cDNA clone encoding the CD98LC was obtained which enabled the mapping of the gene to the long arm of human chromosome 16 (16q24) [17]. The C-terminal amino acid sequence deduced from the nucleotide sequence was identical to the C-terminal sequence of the CD98LC polypeptide purified from MOLT-4 cells [18]. On the other hand, recent studies using expression cloning in Xenopus oocytes have demonstrated that the CD98HC can associate with at least six different human amino acid transporters: hLAT1, hLAT2, hy+LAT1, hy+LAT2, hxCT and hAsc-1 [19], [20], [21], [22], [23], and the CD98LC that was purified from HeLa S3 cells was shown to be identical to hLAT1 [17]. Intriguingly, these amino acid transporters have 12 TM domains, migrate as 40-kDa polypeptides and exhibit amino acid transport activity in the oocytes only when coexpressed with CD98HC. Furthermore, they are phylogenetically very close to one another and the loci for hLAT2 and hy+LAT1 genes are present on the same chromosome, 14q11.2, [20], [21]. Although the loci for hLAT1, hxCT and hAsc-1 genes are distributed to different chromosomes, 16q24, 4q28–q31 and 19q12–q13.1, respectively [17], [22], [23], these amino acid transporters seem to have evolved from a common ancestor [3].

During the course of cDNA cloning of the CD98LC/hLAT1 from HeLa S3 cells, we found mRNA populations whose nucleotide sequences were very similar but not identical to that of CD98LC/hLAT1 (our unpublished observation). This led us to search for a novel amino acid transporter that should be very similar to hLAT1. In the present study, we have cloned and analyzed three types of the hLAT1-like transcripts, suggesting that they do not code for any hLAT1-related amino acid transporters with 12 TM domains but that they are derived from truncated and duplicated paralogs of the hLAT1 gene.

Section snippets

Cells

Human cell lines used include HeLa S3 (cervix carcinoma), MOLT-4 (acute T lymphoblastic leukemia), Jurkat (T-cell leukemia), BALL-1 (acute B lymphoblastic leukemia) and NOMO-1 (acute monocytic leukemia), which were cultured at 37 °C in Eagle’s minimum essential medium (MEM) fortified with 5% calf serum. Human peripheral blood mononuclear cells (PBMC) were isolated from a healthy volunteer’s heparinized whole blood by Ficoll-Hypaque density-gradient centrifugation. PBMC at the interface were

cDNA cloning of hLAT1-related transcripts

Previously, during the course of cDNA cloning of the CD98LC/hLAT1 from HeLa S3 cells, we became aware that there were transcripts whose nucleotide sequences were very similar but not identical to that of hLAT1 (our unpublished observation). Furthermore, a homology search analysis of the hLAT1 nucleotide sequence on the NCBI BLAST (http://www.ncbi.nlm.nih.gov/blast/) revealed that the human chromosome 16p12 BAC clone, CIT987SK-A-61E3 (113916 bp, GenBank accession no. AC003007), harbored a

Discussion

The present study has shown that the three paralogs of the hLAT-1-3TM gene family (IMAA, MMAA and MLAS genes) are transcribed in human PBMC and in various human cell lines to different degrees. The nucleotide sequences of the three types of transcripts are very similar to but distinct from one another and the MMAA and MLAS sequences can be well aligned with the genomic clones CTB-31N19 and RP11-368N21, respectively. The IMAA sequence is well aligned with CTB-31N19 except for its exon 13.

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    The cDNA sequences of IMAA, MLAS and MMAA reported in this paper have been submitted to the DDBJ sequence data bank under accession nos. AB055225, AB055226 and AB055227, respectively.

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